Immunoassays for everolimus

ABSTRACT

Immunoassays for the detection of everolimus are provided. Compounds for producing antibodies for everolimus, as well as antibodies produced therefrom, are also provided.

This application claims priority to U.S. Provisional Application No.60/551,989, filed Mar. 10, 2004, herein incorporated by reference.

FIELD OF INVENTION

The invention relates generally to reagents and methods fordetermination of everolimus in biological fluids.

BACKGROUND OF THE INVENTION

The everolimus [40-O-(2-hydroxyethyl)-rapamycin] is a novel macrolideimmunosuppressant. Everolimus (also known as SDZ-RAD, RAD, Certican®)was developed by Novartis (Nashan B. The role of Certican in the manypathways of chronic rejection. Transplantation Proceedings, 2001, 33:3215-3230, herein incorporated by reference) in an effort to improveupon rapamycin (Sirolimus), a proliferation signal inhibitor that blocksgrowth factor-driven transduction signals in the cellular responses toalloantigen (Cottens S, et al. O-Alkylated rapamycin derivatives andtheir use, particularly as immunosupressants. WO-009409010 28-April-94,herein incorporated by reference). Everolimus has greater stability andenhanced solubility in organic solvents, as well as more favorablepharmokinetics with fewer side effects than rapamycin (Sirolimus).Everolimus has been used in conjunction with microemulsion cyclosporin(Neoral®, Novartis) to increase the efficacy of the immunosuppressiveregime (Kovarik JM, et al. Exposure-response relationship for Certicanin de novo kidney transplantation: define a therapeutic range.Transplantation 2002; 73(6): 920-925, herein incorporated by reference).

The complexity of the clinical state, individual differences insensitivity to immunosuppressive and nephrotoxic effects, has beenrather challenging for physicians to balance between therapeuticefficacy and the occurrence of side effects (Wallemacq, Pierre E.Therapeutic monitoring of immunosuppressant drugs. Where are we?Clinical Chemistry and Laboratory Medicine (2004), 42 (11), 1204-1211,herein incorporated by reference). Therapeutic drug monitoring (TDM),defined as the measurement and interpretation of concentration of thesedrugs in biological fluids, with as a final objective the prediction oforgan responses, became an integral part of transplant protocols.

Therapeutic concentration of everolimus was reported (Kovarik et al.) as3-15 ng/ml, which was consistent with efficacy while minimizing adverseeffects in kidney transplantation. Recent data also showed thattherapeutic drug monitoring (TDM) of everolimus would benefit hearttransplant patients (Starling, Randall C.; et al. Therapeutic drugmonitoring for everolimus in heart transplant recipients based onexposure-effect modeling. American Journal of Transplantation (2004),4(12), 2126-2131, herein incorporated by reference). Everolimus troughlevels were stable in the first year post-transplant, and averaged5.2±3.8 and 9.4±6.3 ng/mL in patients treated with 1.5 and 3 mg/day,respectively.

The TDM of everolimus was reported (McMahon LM, et al. High-throughputanalysis of Certican (RAD001) and cyclosporin A (CsA) in whole blood byliquid chromatography/mass spectrometry using a semi-automated 96-wellsolid-phase extraction system. Rapid Comm. Mass Spectrometry 2000; 14:1965-1971; Brignol N, et al. High-throughput semi-automated 96-wellliquid/liquid extraction and liquid chromatography/mass spectrometricanalysis of Certican (RAD001) and cyclosporin A (CsA) in whole blood.Rapid Communications in Mass Spectrometry 2002; 15: 1-10; Streit F. etal. Rapid liquid chromatography-tandem mass spectrometry routine methodfor simultaneous determination of sirolimus, Certican, tacrolimus, andcyclosporin A in whole blood. Clinical Chemistry. 2002; 48(6): 955-958,each of which herein incorporated by reference). However, methods thatuse HPLC, LC/MS can be impractical for commercial use due to, forexample, long sample preparation time, long assay time, high cost, andlabor-intensive procedures. For routine TDM of everolimus, theavailability of simple automated tests and high throughput clinicalanalyzers is desirable.

SUMMARY OF THE INVENTION

The present invention is directed to novel derivatives of everolimus andnovel everolimus immunogens. The present invention is also directed topolyclonal and monoclonal antibodies generated using everolimusimmunogens, as well as labeled competitors and tracers. Theseantibodies, conjugates, and tracers are useful in immunoassays for thedetection of everolimus in biological fluids.

In one aspect of the invention, competitive immunoassays for determiningthe presence of everolimus in a sample are provided. Illustrativecompetitive immunoassays comprise an antibody capable of specificallybinding everolimus, and an everolimus compound conjugated to adetectable label, wherein the conjugated everolimus compound isconfigured to compete with the everolimus in the sample to bind with theantibody, and wherein the label provides a signal indicative of aconcentration of everolimus in the sample when the everolimus in thesample is present in therapeutic drug monitoring concentrations. In oneembodiment, the immunoassay is suitable for monitoring everolimus in therange of about 3 to about 15 ng/ml. In another embodiment, thecompetitive immunoassay provides a signal indicative of theconcentration of everolimus over a broader range, illustratively fromabout 0 to about 40 ng/ml.

In another aspect of the invention, methods for determining the amountof everolimus in a sample are provided. The methods comprise mixing thesample with an antibody capable of specifically binding everolimus, andan everolimus compound conjugated to a detectable label, wherein theconjugated everolimus compound is configured to compete with theeverolimus in the sample to bind with the antibody, measuring a signalfrom the detectable label indicative of a concentration of everolimus inthe sample, and determining the amount of everolimus in the sample.

In yet another aspect of the invention, compounds are provided havingthe following structure

wherein

-   -   n is 0 or 1;    -   X is a linker chain comprising 3-10 carbon or hetero atoms,        wherein the linker chain may be substituted or unsubstituted and        may be straight or branched;    -   Y is selected from the group consisting of —C(O)—, —NH—, —S—,        —CH₂— and —O—; and    -   Z is an antigenic carrier or a label.        In one particular illustrative embodiment, X is        —CH₂—CH₂—CH₂—CH₂—CH₂—, Y is —C(O)—, and Z is the antigenic        carrier. Antibodies produced using such compounds and        immunoassay kits using the antibodies are also provided.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of an illustrative everolimus derivative ofthe present invention,

FIG. 2 shows synthesis of tethered-RAD-formate,

FIG. 3 shows synthesis of RAD-acid (deprotection),

FIG. 4 shows synthesis of RAD-822 (NHS activation),

FIG. 5 shows synthesis of RAD 822: FAMCO-E FP tracer,

FIG. 6 shows synthesis of RAD 822: BSA immunogen,

FIG. 7 shows the structure of a 32-oxime derivative of everolimus,

FIG. 8 shows an activated ester of the 32-oxime derivative of FIG. 8,

FIG. 9 shows a calibration curve (FPIA) X axis-calibrator values, Yaxis-rates (mp) antibody (polyclonal), FP tracer (RAD822:FAMCO-E),

FIG. 10 shows a comparison of an everolimus FPIA assay according to thepresent invention vs. LC/MS in kidney transplant patients,

FIG. 11 shows a comparison of an everolimus FPIA assay according to thepresent invention vs. LC/MS in heart transplant patients, and

FIG. 12 is an everolimus QMS calibration curve (antibody: polyclonalantibody from RAD 822 immunogen; antigen: Oxime (FIG. 5) derivativecoupled to the particles.

DETAILED DESCRIPTION OF THE INVENTION

“Everolimus” is an immunosuppressive drug, sold by Novartis AG under thetrademark CERTICAN®. Everolimus has the structure of Formula I:

“Haptens” are partial or incompletes antigens. They are usuallyprotein-free substances, mostly of low molecular weight, which are notgenerally capable of stimulating antibody formation, but which do reactwith antibodies. Antibodies may be formed by coupling a hapten to a highmolecular weight antigenic carrier and then injecting this coupledproduct, i.e., immunogen, into a human or animal subject. Everolimus isa hapten.

The phrase “antibody capable of specifically binding everolimus” as usedherein refers to an antibody with the capacity to react with at leastone epitope within the drug in a true antibody-antigen reaction, asopposed to non-specific interaction.

The term “analog” or “derivative” refers to a chemical compound ormolecule made from a parent compound or molecule by one or more chemicalreactions.

An “activated hapten” refers to a hapten derivative that has beenprovided with an available site for reaction, such as by the attachmentof a linking group, for synthesizing a hapten derivative conjugate.

As used herein, a “linking group” or “linker” refers to a portion of achemical structure that connects two or more substructures such ashaptens, carriers, immunogens, labels, tracers, or other linkers. Alinking group has at least one uninterrupted chain of atoms other thanhydrogen (or other monovalent atoms) extending between thesubstructures. The atoms of a linking group and the atoms of a chainwithin a linking group are themselves connected by chemical bonds.Linkers maybe straight or branched, saturated or unsaturated carbonchains. They may also include one or more heteroatoms within the chainor at termini of the chains. By “heteroatoms” it is meant atoms otherthan carbon atoms, illustratively oxygen, nitrogen, sulfur, andphosphorus. The linking group may also include cyclic or aromatic groupsas part of the chain or as a substitution on one of the atoms in thechain. The number of atoms in a linking group or linker is determined bycounting the atoms other than hydrogen. The number of atoms in a chainwithin a linking group is determined by counting the number of atomsother than hydrogen along the shortest route between the substructuresbeing connected. Linking groups may be used to activate a hapten, e.g.provide an available site on a hapten for synthesizing a conjugate of ahapten with a label or carrier.

The terms “immunogen” and “immunogenic” as used herein refer tosubstances capable of producing or generating an immune response in anorganism.

An “active ester” refers to an ester group that can react with a freeamino group of compound such as, for example, peptides and proteins.Examples of active esters include N-hydroxysuccinimide, p-nitrophenyl,pentafluorophenyl, and N-hydroxybenzotriazolyl.

A “carrier” or “immunogenic carrier,” as the terms are used herein, isan immunogentic substance, commonly a protein, that can join with ahapten, thereby enabling the hapten to induce an immune response andelicit the production of antibodies that can bind specifically with theantigen (hapten). Carrier substances include proteins, glycoproteins,complex polysaccharides, particles, and nucleic acid that are recognizedas foreign and thereby elicit an immunologic response from the host.Various proteins may be employed as a poly (amino acid) immunogeniccarrier. These proteins include albumins and serum proteins, e.g.,globulins, ocular lens proteins, lipoproteins, etc. Illustrativeproteins include bovine serum albumin (BSA), keyhole limpet hemocyanin(KLH), egg ovalbumin, bovine gamma-globulin (BGG), etc. Alternatively,synthetic poly (amino acids) may be used. The immunogenic carrier canalso be a polysaccharide, which is a high molecular weight polymer builtup by repeated condensations of monosaccharides. Examples ofpolysaccharides are starches, glycogen, cellulose, carbohydrate gumssuch as gum arabic, agar, and so forth. The polysaccharide can alsocontain poly (amino acid) residues and/or lipid residues. Theimmunogenic carrier can also be a poly (nucleic acid) either alone orconjugate to one of the poly (amino acid)s or polysaccharides mentionedabove. The immunogenic carrier can also be a particle. The particles areillustratively at least about 0.02 microns (μm) and illustratively notmore than about 100 μm, and usually about 0.05 μm to 10 μm in diameter.The particle can be organic or inorganic, swellable or non-swellable,porous or non-porous, optionally of a density approximating water,generally from about 0.5 to 1.5 g/ml, and composed of material that canbe transparent, partially transparent, or opaque. The particles can bebiological materials such as cells and microorganisms, includingnon-limiting examples such as erythrocytes, leukocytes, lymphocytes,hybridomas, Streptococcus, Staphylococcus aureus, E. coli, and viruses.The particles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

“Poly (amino acid)” or “polypeptide” is a polyamide formed from aminoacids. Poly (amino acid)s will illustratively range from about 2,000molecular weight, having no upper molecular weight limit, normally beingless than 10,000,000 and optionally not more than about 600,000 daltons.There will usually be different ranges, depending on whether animmunogenic carrier or an enzyme is involved.

A “peptide” is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of a-amino acids inwhich α-amino group of each amino acid residue (except the NH2 terminus)is linked to the α-carboxyl group of the next residue in a linear chain.The terms “peptide”, “polypeptide”, and “poly (amino acid)” are usedsynonymously herein to refer to this class of compounds withoutrestriction as to size. Larger members of this class are also referredto as proteins.

A “label”, “detector molecule”, or “tracer” is any molecule thatproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, an immunogen, an antibody,illustratively the antibody produced in response to the antigeniccompound or a secondary antibody having specificity therefor, or toanother molecule such as a receptor or a molecule that can bind to areceptor such as a ligand, particularly a hapten. Non-limiting examplesof labels include radioactive isotopes, enzymes, enzyme fragments,enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores, dyes, chemiluminescers, luminescers, sensitizers,non-magnetic or magnetic particles, solid supports, liposomes, ligands,receptors, and hapten radioactive isotopes.

The term “antigenic compound” as used herein is a compound used toproduce an immune response. Illustratively, the antigenic compound is ahapten, for example everolimus, linked to an immunogenic carrier. Theantigenic compound is used to generate the desired antibodies.

The term “labeled competitor” as used herein is a molecule capable ofspecific binding to antibodies having specificity for everolimus,wherein the molecule is linked to a detectable label or tracer.Illustratively, the molecule is everolimus or a derivative or analytethereof.

The term “biological sample” includes, but not limited to, any quantityof a substance from a living thing or formerly living thing. Such livingthings include, but are not limited to, humans, mice, monkeys, rats,rabbits, horses, and other animals. Such substance include, but are notlimited to, blood, serum, urine, tears, cells, organs, tissues, bone,bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovialmacrophages, endothelial cells, and skin.

The term “therapeutic drug monitoring concentrations” refers toconcentrations of the drug from that which provides no effect up to thatof toxic effect. For everolimus, as is currently understood, thetherapeutic range is generally 3-15 ng/ml. However, it is understoodthat it may be useful to provide information across a broader range, andthe assay range is generally broader than the therapeutic range.Accordingly, assays that monitor therapeutic drug monitoringconcentrations may provide sensitivity across a broader range ofeverolimus concentrations.

The term “patient” includes human and animal subjects.

Numerous quantitative immunoassay formats for detecting a hapten such asa drug or other small molecule in a body fluid are known. An assaymethod for everolimus illustratively includes combining the sample withan anti-everolimus antibody and detecting the amount of theanti-everolimus antibody-everolimus complex, as indicative of the amountof everolimus in the sample.

Illustrative immunoassays employ polyclonal antibodies and/or monoclonalantibodies with appropriate sensitivity and specificity to everolimus toprovide information about everolimus concentrations statisticallycomparable to that obtained through analytical methods such as LC/MS.Such immunoassays illustratively are useful in monitoring levels of thedrug in patient samples.

Designing an immunoassay for the detection of a small molecule such as adrug can be a challenge. Such small molecules often lack antigenicity,making it difficult to generate antibodies. This is particularlyproblematic with drugs such as everolimus, which suppress the immuneresponse. To increase the immunogenicity, larger antigenic compounds,illustratively proteins or polypeptides, including but not limited tobovine serum albumin, ovalbumin, keyhole limpet hemocyanin, and thelike, are conjugated to the drug. Further, detection of the drug in animmunoassay generally requires the use of a detectable label conjugatedto an antibody, an analyte, or analyte analog.

Immunogens may be made by coupling everolimus to an antigenic carrierprotein through a linker reacted with one of the hydroxy groups,illustratively the hydroxyl at position 28. Such methods are describedin U.S. Pat. No. 6,635,745 and European Patent No. EP 0 693 132, hereinincorporated by reference. However, it has been found that an extendedlinker between the antigenic carrier leads to the production of moresensitive antibodies. Without being bound to any particular theory,presumably, the longer linker provides for a more accessible epitope,resulting in increased specificity of the antibody for everolimus.

In one illustrative example, the immunogenic conjugate is illustrativelya compound shown in FIG. 1 and in Formula II below:

wherein

-   -   n is 0 or 1;    -   X is a linker chain comprising 3-10 carbon or hetero atoms,        wherein the linker chain may be substituted or unsubstituted and        may be straight or branched;    -   Y is selected from the group consisting of carbonyl, —NH—, —S—,        —CH₂— and —O—; and    -   Z is an antigenic carrier.        In this embodiment, the linker (X) comprises a chain of three or        more atoms, wherein at least one atom is carbon (C). The linker        molecule can be a straight or branched chain and, in additional        to at least one C atom, can contain heteroatoms such as N, O, S,        and P, which can be substituted independently of one another.        The linker can also contain multiple bonds. If the linker is        branched, the branches may form rings. When the linker is        branched, the length of the linker is determined by the number        of atoms in the shortest path between the everolimus and the        conjugate. Illustratively, the linker (X) comprises a chain of        between three and ten atoms, more illustratively between four        and seven atoms, and even more illustratively four to five        atoms. In one particularly well suited example, the linker        comprises a chain of five atoms. It is understood that antigenic        compounds or other everolimus conjugates can be made by coupling        through other positions on the everolimus molecule,        illustratively one of the ketone groups at positions 26, 32, or        9, by way of suitable linkage (e.g. oxime, hydrazone), wherein        the linker has sufficient length to provide access to a suitable        everolimus epitope, while not providing too much separation        between the everolimus and the antigenic carrier. The linker may        be appended to a suitable functional group that permits coupling        to a protein or other biomolecule, or to a solid support        surface.

In one particular example, n=1, X is a chain of 5 atoms, and Y is—C(O)—. An example of such an immunogenic compound is RAD 822:BSA, asshown in FIG. 6, wherein Z is BSA. Antibodies produced using RAD 822:BSAhave proven to demonstrate strong binding (>100 mP) and stronginhibition (>50% @ 50 ng/mL), as shown in the calibration curve of FIG.9. Another such example wherein strong binding and strong inhibition isexpected is antibodies produced using an immunogenic compound of FormulaII wherein n=1, X is a chain of 7 atoms (for example —CH₂)₇—), and Y is—C(O)—. It is expected that antibodies produced using other compounds ofFormula II will also demonstrate strong binding and strong inhibition.It is understood, however, that medium binding (50-100 mP and/or mediuminhibition (50% at 50-500 ng/mL) may be acceptable for some embodiments.The antibodies generated from the compounds of Formula II are wellsuited for competitive and non-competitive assays.

The immunogenic conjugate is useful for generation of polyclonal as wellas monoclonal antibodies. Depending upon the purpose of the detectionsystem, antibodies may be selected to target everolimus with little orno cross-reactivity to metabolites, or, alternatively, antibodies withthe capacity to recognize one or more of the metabolites and/or relateddrugs as well as everolimus can be selected. A comprehensive study ofthe biotransformation of everolimus by human liver microsomes hasidentified at least 11 metabolites resulting from hydroxylation anddemethylation of everolimus (Bomsen KO, et al. Electrospray ionizationand collisionally induced dissociation of RAD001 and related compoundsand structural characterization of RAD001 metabolites by nano-spray andmicro liquid chromatography mass spectrometry, Jacobson W, et al.Comparison of the in vitro metabolism of the macrolideimmunosuppressants sirolimu and RAD. Transplantation Proceedings 2001;33: 614-615, herein incorporated by reference), with the majormetabolites being hydroxyl-(24/25 OH RAD, 460H RAD), ring-open compounds(RAD SA, RAD PSA), and 40-phosphatidylcholine-RAD (RAD PC). Thegeneration of everolimus metabolites is attributed to cytochrome P4503A4, the most abundant of the CYP enzymes in the liver and intestine,also involved in Cyclosporin (CsA) and rapamycin metabolism.Illustratively, if antibodies capable of distinguishing everolimus frommetabolites are desired, antibodies optionally may be induced byimmunogens made from 28-O-derivatives.

Besides immunogens, other everolimus conjugates may be prepared. Whenthe linkage via the 28-O-position is used to produce a labeledcompetitor molecule, Z of Formula II may be biotin, horseradishperoxidase or other enzymes, fluorescent labels, and dyes, or particlessuch as metal sols, latex particles, polystyrene particles and the like,or any other label, detector molecule, or tracer, as discussed above.Such conjugates are formed by any number of routine procedures wellknown to those skilled in the art. It is possible to prepare conjugatesof everolimus that are useful for a variety of immunoassays, includingbut not limited to fluorescence polarization immunoassay, cloned enzymedonor immunoassay, lateral flow immunoassays, chemiluminescencemicroparticle assays and immunoturbidimetric assays. Several embodimentsare described herein.

In one illustrative embodiment, antibodies are produced using anantigenic compound of Formula I, for example RAD 822:BSA, as shown inFIG. 6. In one competitive assay, the labeled competitor may be derivedfrom everolimus using a linkage that is the same as or similar to thatof the antigenic compound, for example RAD 822:FAMCO-E, as shown in FIG.5. However, it is understood that when the labeled competitor is a 28-O—derivative of everolimus, the linker chain, as represented by X inFormula II, is not limited to a chain comprising 3-10 atoms. In somecompetitive assays, it is desirable to have a labeled competitor thatbinds to the antibody with less specificity than everolimus binds to theantibody, allowing the labeled competitor to be displaced more readilyin the presence of everolimus. Accordingly, shorter linkers may bedesirable, to limit access of the antibody binding sites and to weakenspecific binding with the antibody. Linkers longer than 10 atoms may beuseful as well, as the distance between the label and the everolimus isnot critical in many embodiments. Thus, it is understood that the linkerof the labeled competitor is not limited to the definition of X inFormula II.

In another example, the labeled competitor is derived from everolimususing a different linkage. One suitable labeled competitor, as shown inFormula III:

wherein

-   -   X¹ is a linker chain comprising one or more atoms, each of which        may be substituted or unsubstituted and may be branched or        unbranched;    -   Y is defined as above; and    -   Z is a label, as defined above.        For a labeled competitor, the structure of the linker X¹ may be        chosen based on the particular assay. As discussed above, it may        be desirable to provide a short linker having only one or two        atoms in the chain (illustratively —O—CH₂—), to reduce specific        binding to the antibody. On the other hand, it may be desirable        to provide a long linker, illustratively when the everolimus of        the labeled competitor is being tethered to a solid support, and        additional flexibility in the linker chain is desired. The        longer linker may comprise a chain of any length, illustratively        from 10 to 100 atoms. Further, it is understood that a linker as        defined as X above, is suitable in many applications. Still        further, it is understood that compounds of Formula III, wherein    -   X¹ is X, as defined above for Formula II;    -   Y is defined as above Formula II; and    -   Z is an antigenic carrier, are suitable antigenic compounds for        producing antibodies for everolimus.

One illustrative competitive assay uses antibodies produced using anantigenic compound of Formula II, illustratively RAD 822:BSA, as shownin FIG. 6, and a labeled competitor of Formula III, illustratively the32-oxime compound shown in FIG. 7, wherein the succinimide is replacedwith a suitable label for a competitive immunoassay. For FPIA, the labelmay be FAMCO-E, although other suitable labels or tracers may be used.Since the unbound drug-everolimus has higher affinity for the antibody(produced from RAD 822:BSA immunogen) than the labeled 32-oximederivative, a very small quantity of unbound everolimus in a sampleshould have detectable effect on the rate of agglutination governed bythe bound oxime derivative. Thus, if the labeled 32-oxime derivative is,for example, immobilized on latex, a high sensitivity (required fordetection of everolimus due to the extremely low and narrow therapeuticrange-3-15 ng/ml) can be achieved.

Oxime linkages are more stable to hydrolytic cleavage than ester bonds.In addition, it has been found that the oxime derivative (position 32)will not undergo an elimination reaction in most useful conditions. Theoxime derivate (activated ester) would be a desirable candidate invarious assays, including latex enhanced immunoturbidimetric assays.Micro-particles coupled the oxime derivative have showed improvedstability.

It is understood that any combination of antibodies produced using theabove-described antigenic compounds and the above-described labeledcompetitors may be used in competitive assays, the choice of whichdepend on the specific assay and desired sensitivity.

Fluorescence Polarization Immunoassay for Everolimus

Fluorescence polarization immunoassay (FPIA) technology is based uponcompetitive binding between an antigen/drug in a sample and a knownconcentration of labeled antigen/drug. FPIA is described in U.S. Pat.No. 4,593,089, incorporated herein in its entirety. In the assay system,the sample antigen, such as everolimus, competes withfluorescein-labeled antigen or antigen-analog for a fixed number ofantibody sites. The main components of the FPIA system are: i) antibodycapable of specifically binding to the antigen/drug, ii) the samplesuspected of containing the antigen/drug, and iii) the antigen/drug oranalog labeled with fluorescein. Because of the rotational properties ofmolecules in solution, the degree of polarization is directlyproportional to the size of the molecule. Polarization increases asmolecular size increases. When linearly polarized light is used toexcite the fluorescein-labeled antigen/drug, which is small and rotatesrapidly in solution, emitted light is significantly depolarized. Whenfluorescein-labeled antigen/drug is bound to antibody, rotation isslowed and emitted light is highly polarized. Increased amounts ofunlabeled antigen/drug in the sample will result in decreased binding offluorescein-labeled antigen/drug by antibody, and decreased polarizationof emitted light from sample. In the present examples, the preciserelationship between polarization and concentration of the unlabeledeverolimus in the sample is established by measuring the polarizationvalues of calibrators with known concentration of everolimus.

Homogeneous Microparticle (Immunoturbidimetric) Immunoassay forEverolimus

Format A:

In one embodiment, a kit is provided with a liquid reagent set used forperforming immunoturbidimetric assays for the measurement of everolimusconcentrations in whole blood, blood hemolysate, serum or plasma. Inthis technology, an everolimus conjugate, illustratively a28-O-activated everolimus conjugate, is loaded on a microparticle, forexample, any of the microparticles manufactured and/or sold by Seradyn,Inc. (Indianapolis, Ind.), including, but limited to, polystyrene orcarboxylate-modified polystyrene and streptavidin-coated magneticparticles. Antibody capable of specifically binding everolimus isformulated in a standard buffer system. A competitive reaction takesplace between everolimus immobilized on the microparticles andeverolimus in the patient's sample for binding to a limited amount ofanti-everolimus antibody in the reaction solution. Agglutination ofparticles is inhibited by the presence of drug in patient sample.

Format B:

This embodiment is similar to that described above as Format A exceptthat an antibody capable of specifically binding everolimus is loaded onthe microparticle. A derivative of everolimus, illustratively28-O-activated everolimus, is linked to a macromolecule of choice, forexample, bovine serum albumin, ovalbumin, dextran, and the like, to forma drug conjugate. A competitive reaction takes place between the drugconjugate in buffered solution and everolimus in the patient's samplefor binding to anti-everolimus antibody immobilized on themicroparticles. Agglutination of particles is inhibited by the presenceof drug in patient sample.

Cloned Enzyme Donor Immunoassay-CEDIA® technology for Everolimus

CEDIA® (trademark of Roche) has proven to be a highly accurate methodfor quantitation of therapeutic drugs. CEDIA® is the subject of severalpatents including U.S. Pat. No. 4,708,929, claiming competitivehomogeneous assay methods, U.S. Pat. No. 5,120,653, claiming arecombinant DNA sequence for coding the enzyme donor fragment and a hostfor such a vector, U.S. Pat. No. 5,604,091, claiming amino acidsequences of the enzyme donor fragment, and U.S. Pat. No. 5,643,734,teaching kits for CEDIA® assays. All of the above patents are hereinincorporated by reference in their entirety. CEDIA is based upon thecompetition of a drug in the biological sample with drug conjugated tothe inactive genetically-engineered enzyme-donor (ED) fragment fromβ-D-galactoside galactohydrolase (E.C. 3.2.1.23) or B-galactosidase (βgal) from E. coli for binding to an antibody capable of specificallybinding the target drug. If the target drug is present in the sample, itbinds to the antibody, leaving the ED portion of the ED-drug conjugatefree to restore enzyme activity upon association with enzyme acceptor(EA) fragments, also from B-D-galactoside galactohydrolase (E.C.3.2.1.23) or β-galactosidase (β gal) from E. coli, in the assay reactionmixture. The active enzyme is then capable of producing a quantifiablereaction product when exposed to appropriate substrate. An illustrativesubstrate is chlorophenol red-β-D-galactopyranoside (CPRG), cleaved bythe active enzyme into galactose and CPR. CPR is measured by absorbencyat wavelength 570 nm. If drug is not present in the sample, the antibodybinds to the ED-drug conjugate, inhibiting association of the EDfragments with the EA fragments, thus inhibiting restoration of enzymeactivity. The amount of reaction product and resultant absorbance changeare proportional to the amount of drug in the sample.

Chemiluminescence Heterogeneous Immunoassay

In one embodiment, a competitive assay using chemiluminescencemicroparticle immunoassay (CMIA) technology comprises use of antibodies,capable of specifically binding to everolimus, coupled to particles, inparticular magnetic particles or particles suitable for separation byfiltration, sedimentation or other means. A label comprising everolimuslinked to a suitable chemiluminescent molecule, for example anacridinium ester, competes with free everolimus in the patient's samplefor the limited amount of anti-everolimus antibody on the magneticparticle. After a routine wash step to remove unbound label, the amountof chemiluminescence, expressed in Relative Light Units (RLU), ismeasured. The amount of chemiluminescence is inversely related to theamount of free drug in the patient's sample and concentration isdetermined by constructing a standard curve using known values of thedrug.

Other Immunoassay Formats

The derivatives, antibodies, immunogens, and/or other conjugatesdescribed herein are also suitable for use with any of a number of otherhomogeneous and heterogeneous immunoassays with a range of detectionsystems. The examples presented herein are not intended to be limiting.

Thus, the present invention provides everolimus derivatives that areuseful for the preparation of immunogens and conjugates for use inimmunoassays for the detection of everolimus. By coupling an everolimusanalog according to the present invention to an immunogentic carriermaterial, polyclonal or monoclonal antibodies can be produced andisolated, which are useful reagents for immunoassays for the detectionof everolimus.

Coupling can be accomplished by any chemical reaction that will bind thelabel or carrier. This linkage can be accomplished by a variety ofchemical mechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding, and complexation. Most often thelinkage is made through covalent bonding. Covalent binding can beachieved either by direct condensation of existing side chains or byincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas a carrier, to other molecules. Representative coupling agents includeorganic compounds such as thioesters, carbodiimides,N-hydroxysuccinimide esters, diisocyanates, glutaraldehyde,diazobenzenes, and hexamethylene diamines. It is understood that thislisting is not an exhaustive compilation of the various classes ofcoupling agents known in the art but, rather, is representative of themore common coupling agents.

Illustrative everolimus immunoassays employ anti-everolimus antibodiesthat can be either polyclonal or monoclonal. In illustrative competitiveimmunoassays, the antibody preparation used is induced by an immunogendescribed herein is formulated in an aqueous solution such as buffer,and the like or provided in an adjuvant or similar composition. Theinduced antibodies can be tested to determine specificity foreverolimus.

EXAMPLE 1 Synthesis of Immunogenic Compounds and Labeled CompetitorsSynthesis of RAD-mono-formate

0.6 g of RAD was dissolved in 2 mL of dry methylene chloride in a 100 mLround bottom flask under an atmosphere of argon (Ar). An aliquot (˜10μL) was saved for HPLC and TLC assays. The round bottom flask containingthe reaction solution was placed in the ice/NaCl bath at about −20° C.The reaction flask was allowed to cool down for 3-5 min. Dry pyridine(0.25 mL) was added using a dry glass syringe with a metal needle (15cm) all at once. 0.35 mL dry allyl chloroformate was added using a dryglass syringe with a metal needle (15 cm) within about ½-1 min. Shortlyafter addition, precipitation occurred. The stirring was allowed for onehour. The reaction was quenched by adding 5 mL of saturated NaHCO₃. Thequenched reaction was extracted with methylene chloride. The organicphase was combined, dried (Na₂SO₄) and filtered. The filtrate wastransferred to a 250 ml bottom flask (100-250 mL) and the volatiles wereevaporated under reduced pressure (water bath below +30° C.). The crudeproduct was purified in silica chromatography in 40% ethyl acetate inmethylene chloride. The final product was yielded 0.53 g.

Synthesis of Tethered-RAD-Formate (FIG. 1)

RAD-monoformate 0.4 g, DMAP 9 mg, pimelic acid mono allylester 0.2 gwere placed in a dry 50 mL round bottom flask equipped with a stir bar.5 mL of dry CH₂Cl₂ was added to the above flask that is placed in anice/water bath at 0° C. under Argon. The solution was allowed to coolfor 5-10 min. DCC 0.2 g was added quickly. The reaction was allowed tostir for five hours at 0° C. The precipitated DCU was collected on aWhatman #1 filter. The flask and precipitate were rinsed with about 10mL of ice cold CH₂Cl₂ combined, and the organic layer was washed withice cold 1M HCl, saturated aqueous sodium bicarbonate, dried overNa₂SO₄, decanted (or filtered), the solid rinsed with 2×5 mL CH₂Cl₂, andthe crude product concentrated under vacuum yielding 0.45 g. Furtherpurification can be achieved by flash chromatography on silica gelcolumn in ethyl acetate/methylene chroloride (1:1).

Synthesis of RAD-Acid (Deprotection) (FIG. 2)

0.362 g of protected RAD was placed under argon in dry amber roundbottom flask equipped with a stir bar and a rubber seal at roomtemperature. The reagents were allowed to warm for at least 30-min atroom temperature to protect from moisture condensation. 5.75 mL of drymethylene chloride was added into the reaction flask using a dry syringe(plastic or glass) with a dry needle and 73 μL (4 equivalents) ofglacial acetic acid was added using an automatic pipette.Tetrakis-(triphenylphosphine)-palladium(0) (Pd(PPh₃)₄) 7.4 mg (0.02equivalent or 2%) was weighted out on a weighing paper and added to thereaction solution. 260 μL of Tributyltin hydride was added drop-wise tothe above reaction mixtures at room temperature. The reaction wasallowed to stir for about 30 min at room temperature. The flask wasplaced on a rotary evaporator and condensed the content to about 2-3 mLsolution. The solution was loaded on a short 5-6 cm glass chromatographycolumn (dry SiO2 volume=50 cm³, diameter about 4 cm). Elute: 1^(st)-200mL ethyl acetate, 2^(nd)-200 mL 10% acetone in ethyl acetate, 3^(rd)-200mL 50% acetone in ethyl acetate, pure acetone about 1.5 L. The productfractions were collected and concentrated under vacuum yielding 273 mgof RAD acid.

Synthesis of RAD-822 (NHS activation) (FIG. 3)

RAD-acid 0.281 g obtained in the previous step was allowed to warm toroom temperature and back filled with argon. DMAP 3.1 mg (0.1equivalent), N-hydroxysuccinimide (NHS) 88 mg (3 equivalents), DCC 79 mg(1.5 equivalent) were added quickly into the reaction flask. Thereaction was initiated by adding 3.0 mL of dry methylene chloride viasyringe. The reaction was allowed to proceed at room temperature for 1hr stirring under argon. The reaction was placed on an ice/water bathand allowed to stir for additional 2 hrs. At the end of this time 1.5 mLof hexane was added and stirring was stopped. The precipitated urea(DCU) was filtered through a cotton plug using a Pasteur pipette. Theorganic phase was extracted in sequence with equal volumes of ice cold1.0M HCl, sat. NaCl, sat. NaHCO₃, sat. NaCl, distilled water. Theorganic phase was dried with Na₂SO₄, and concentrated under reducedpressure. The crude RAD-822 was dissolved in about 1 mL of CH₂Cl₂ andloaded on the silica gel column. The sample was eluted by hexane/acetone(1:1). The product was collected and concentrated under vacuum yielding46 mg.

RAD-822 may be used in various embodiments of the present invention.This compound is used herein in an illustrative FPIA embodiment with anester modified linker at position 28. However, RAD 822 can undergochemical degradation via hydrolytic cleavage due to an eliminationreaction. Thus, RAD 822 is not optimal for other embodiments requiringvigorous heat stressing conditions, such as QMS® technology (Seradyn,Indianapolis, Ind.).

Synthesis of RAD 822: FAMCO-E FP tracer (FIG. 4)

A 100 ml round bottom flask was weighed and 1 ml solution of FAMCO-Esolution was pipetted into a suitable sized round bottom flask. Thesolvent was rotavapored off under reduced pressure. The flask withplastic cap was wrapped around with aluminum foil and a magnetic stirrerwas added to the above flask. 1 ml DMF solution was pipetted into theflask above and stirred at room temperature for 40-50 minutes. Asufficient quantity of RAD was removed from −78° C. freezer storage andplaced at room temperature to thaw. 4 mg of RAD 822 was weighed onto apiece of weighing paper and the powder transferred to the flaskcontaining FAMCO-E solution. The reaction was allowed to stir underArgon pressure on the magnetic stir plate for 1 hr in an ice/water bathand then 30-40 minutes at room temperature (no more than 2 hours total).The solvent was evaporated under reduced pressure (high vacuum pump).The crude product (0.5-2 ml for a 1 mg batch size) was dissolved inminimal amount of methanol. TLC Solvent was prepared by adding 85 mlCH₂CL₂ and 1.5 ml MeOH. The plates were allowed to air dry for at least60 minutes in an operating fume hood, and the tracer band was scrapedfrom the plate. The powder was transferred to a 30 ml (porosity M)Buchner filter funnel and washed with 100% methanol. Filtrate wascollected and transferred to a suitable sized round bottom flask. Thefiltrate was concentrated to dryness and re-dissolved in methanol. Thesolution was filtered through a 0.45 μm filter and the filtrate wascollected in the amber vessel.

Synthesis of RAD 822: BSA Immunogen (FIG. 5)

In a 100 ml round bottom flask equipped with a teflon-coated magneticstir bar, the DMSO (6.8 ml) solution of RAD 822 (27.2 mg) was addedslowly drop wise to BSA: PBS (10 ml, 20 mM PBS pH 7.2) solution undervigorous stirring conditions at room temperature. The solution startedturning cloudy gradually. The round bottom flask was covered withaluminum foil and allowed to stir for another 2 hours at roomtemperature, then it was dialyzed in a snake skin dialysis tube (Pierce)against 50 mM PBS buffer pH 7.5 in a cold room five times. Final volumewas 45 mL. The solution was concentrated to 9 mL (˜8 mg/mL) using AmiconCentriprep concentrator (Lot 874710, 10 kD MWCO). The solution was mixedwell to assure homogeneity.

Synthesis of Everolimus-O-carboxymethyl-32-oxime (FIG. 6)

To a solution of everolimus, 290 mg in 3.0 mL of dry pyridine at +23°C., was added 160 mg of carboxymethoxylamine hemihydrochloride. Thereaction was conducted under an inert atmosphere in a round bottom flaskequipped with a stir bar. After stirring the reaction solution for 5-6hours, the content was diluted with ˜25 mL of methylene chloride andextracted successively with equal volumes of 1.2 M cold HCl, saturatedsodium bicarbonate, and saturated sodium chloride. The resulting organicphase was dried with anhydrous sodium sulfate, concentrated under vacuumand used in the next activation step as is. HPLC analysis on “Silica”stationary phase (from Regis Technologies) using 4% methanol, 40% ethylacetate in hexane as a mobile phase at 2 ml/min (UV detection at 280 nm)indicates that the reaction leads to formation of isomers (E and Z), andthe ratio is 3:1 (assuming similar extinction coefficients). Exact MolMass: 1030.6. MS-ESI (M+Na⁺): 1052.8.

Synthesis of an Activated Ester (Succinimide) ofEverolimus-O-carboxymethyl-32-oxime (FIG. 7)

Dry N,N-dimethylamino pyridine (DMAP) 7 mg, dicyclohexylcarbodiimide(DCC) 124 mg, N-hydroxysuccinimide (NHS) 173 mg andEverolimus-O-carboxymethyl-27-oxime 309 mg (from the previous step) allwere mixed together dissolved in dry methylene chloride and chilled to0° C. After stirring for about 6 hours under inert atmosphere, theresulting suspension was filtered and extracted consecutively with 1.2 MHCl, saturated sodium bicarbonate, and saturated sodium chloride. Theresulting organic phase was dried with anhydrous sodium sulfate andchromatographed on silica gel using successively the following solventmixtures: hexane/acetone (3/2), hexane/acetone (1/1), hexane/acetone(2/3) all v/v. Major product-containing fractions were combined andconcentrated under vacuum yielding 116 mg of the activated ester.

HPLC analysis on “Silica” stationary phase (from Regis Technologies)using 4% methanol, 40% ethyl acetate in hexane as a mobile phase at 2ml/min (UV detection at 280 nm) indicates that the reaction leads toformation of E and Z isomers (no base line resolution). Thin layerchromatography in mixture of acetone/hexane (3/2, v/v): Rf-0.48. ExactMol Mass: 1127.6. MS-ESI (M+Na⁺): 1150.6.

The resulting compound is shown in FIG. 8. It is understood that thesuccinimide group may be replaced with an immunogen, similar to theprocess described above with RAD 822 to obtain RAD 822:BSA, or thesuccinimide group may be replaced with a label, similar to the processdescribed above with RAD 822 to obtain RAD 822:FAMCO-E. It is understoodthat other immunogens, labels, and tracers may be used.

EXAMPLE II Antibody Preparation

Polyclonal anti-everolimus antibodies can be prepared by conventionalmethods. Animals were immunized with everolimus immunogen (RAD 822/BSA),as produced in Example I. The immunization program started with initialinjection of 0.5 ml immunogen mixing with 0.5 complete Freunds adjuvant.Subsequent injections were performed with 0.5 ml immunogen mixing with0.5 incomplete Freunds adjuvant. Animals were typically injected everytwo weeks. Sera were screened via FPIA using RAD 822: FAMCO-E tracer.Several bi-monthly production bleeds (˜20 mL per bleed) from threerabbits were pooled together. Before filter and dilution, the totalpooled volume is about 500 mL. Anti-Sera from Rabbit were filtered with0.2 um Cellulose Acetate Filter under vacuum and diluted with phosphatebuffer with sodium azide and sodium chloride at pH 7.5. The final volumeis about 1000 ml.

Monoclonal anti-everolimus antibodies can be prepared by immunization ofmice. A mouse can be injected with a composition containing an immunogenof this invention and Freund's adjuvant. After the last immunization,the mouse was killed and spleen was processed. The spleen cells werefused with myeloma cells. The fused cells were allowed to grow andsupernatant was screened via ELISA.

EXAMPLE III Fluorescence Polarization Immunoassay using RAD 822: FAMCO-Etracer Automated Fluorescence Polarization Immunoassay (FPIA)

This example describes an exemplary fluorescence polarizationimmunoassay (FPIA) for everolimus.

The fluorescence polarization immunoassay was performed using anautomated TDx polarization analyzer (Abbott Laboratories, Irving, Tex.)using a competitive assay included anti-analyte antibody (theanti-everolimus antibody of Example II) or “A”, a fluorescein:everolimusanalog conjugate (tracer or “T”), and a pretreatment buffer or “B.” Thecalibration of the automated assay described in the examples wasachieved with a series of six calibrators that include specifiedconcentrations of everolimus spiked into human serum. Patient samples(plasma) are placed in plastic sample cups in a circular carouseldesigned for the TDx instrument. The automated assay is described indetail in literature available from Abbott Laboratories, Irving, Tex. Itis understood that, while the TDx polarization analyzer is used in thisexample, other devices may be used to detect the polarization in anFPIA.

Specimen Collection and Preparation for Analysis

This assay has only been characterized for trough samples.

Whole blood treated with EDTA is used for each assay. Each assay uses600 μl of whole blood, collected using normal aseptic venipuncturetechnique in glass or plastic EDTA tubes. Illustratively, patientsamples can be stored at 2-8° C. for up to 24 hours. If longer storageis required, whole blood may be frozen at −20° C. or colder and can betested up to 28 days later. Illustratively, frozen samples are thawedcompletely and mixed thoroughly prior to use. All samples (frozen andfresh) are mixed thoroughly by gently inverting multiple times prior toperforming the sample extraction.

Reagents

Antibody Reagent (5 mL)—<5% rabbit antisera in buffer containing proteinas stabilizer and <0.1% sodium azide as preservative. The polyclonalAntibody Reagent was produced in methods consistent with Example II,using the immunogen shown in FIG. 6 (RAD 822:BSA).

Tracer Reagent (5 mL)—<1% fluorescein tracer (RAD 822: FAMCO-E FP, asshown in FIG. 5) in buffer containing 0.01 M PBS, pH 7.5, 0.1% sodiumazide, and 0.01 mg/ml bovine gamma globulin. Vial cap labeled “T”.

Pretreatment Buffer (5 mL)—tris buffer, detergent, and <0.1% sodiumazide as preservative. Vial cap labeled “B”.

Precipitation Reagent. (7.5 mL)—containing precipitating reagent and<0.1% sodium azide.

Assay Procedure

A. Sample Extraction Procedure:

Samples (calibrators, patient samples, and controls) are extracted justprior to analysis by the instrument. 600 μl of each calibrator, control,or sample to be assayed is pipetted into the appropriate centrifugetube. 700 μl of methanol is dispensed to the sample and 100 μl ofPrecipitation Reagent is added into each centrifuge tube containingsample and methanol. Each centrifuge tube is capped immediately toprevent evaporation, then mixed/vortexed vigorously at the highest speedfor at least 10 seconds, and centrifuged for at least 8 minutes at13,400×g. After centrifuging, at least 300 μl of each supernatant ispipetted into sample cartridges loaded into the carousel and theinstrument is started immediately, to minimize sample evaporation.

B. Summary of Procedure:

Two cartridges and 2 cuvettes for each patient sample and control areplaced into an assay carousel. At least 300 μl of the supernatant fromSample Extraction Procedure is pipetted into each sample well, avoidingbubbles. The reagents are mixed by gentle inversion. The vial caps areremoved and the reagent set and the assay carousel are placed in theanalyzer and the assay process started without delay.

The reference material used was prepared by gravimetric addition tohuman blood hemolysate. Each lot of calibrator is value assigned on theTDx® using a reference material that is traceable to a validated LC/MSmethod. As currently configured, the assigned value concentration ofeach level of calibrator is printed on the calibrator carton label andenclosed calibrator value card. These values are programmed into theTDx® parameters with each new lot of calibrator. The assay range is from2.00 ng/nl to the assigned value of the F calibrator (˜40 ng/1 nL).

Results

The illustrative assay system is a quantitative procedure. Everolimusconcentrations are recorded on the TDx®/TDxFlx® analyzer printout inng/mL. Patient sample results lower than the sensitivity of the assayshould be reported as “<2.00 ng/mL”. The everolimus concentration inmost samples will fall within the assay range. If the value from apatient sample is greater than the F calibrator, “HI” will be printed.Illustratively, such samples may be manually diluted (1:1 or 1:4) withwhole blood negative for everolimus, re-assayed by perform the sampleextraction procedure, and the final printed value multiplied by thedilution factor to obtain the true concentration.

It is preferred that calibrators, controls, and samples should be run inreplicates of two or greater and the mean value reported. It ispreferred that results with a coefficient of variation (CV) ofduplicates greater than 20% be repeated.

The majority of the sample extract is methanol. Due to the volatility ofmethanol, the time between extraction and sample analysis has beenlimited to prevent evaporation. Evaporation of the samples may lead tofalsely elevated results. Accordingly, if samples are loaded onto theinstrument and the run is aborted, these samples have been re-extractedand re-run.

C. Performance Characteristics of the FPIA using Polyclonal AntibodyReagent (RAD 822:BSA) and Tracer Reagent (RAD 822:FAMCO-E)

1. Recovery of Spiked Samples

Whole blood specimens negative for everolimus were spiked witheverolimus across the assay range then assayed n—3 on three differentanalyzers. The mean value was compared to LC/MS value, and percentrecovery calculated. The results are shown in Table 1. TABLE 1 Percentrecovery = mean TDx result ÷ LC/MS result × 100% TDx LC/MS Percent AvgTDx Result (ng/mL) Avg LC/MS Result (ng/mL) Recovery 32.07 39.53 81%22.32 28.46 78% 14.52 17.39 83% 7.27 9.49 77% 3.63 4.74 77%

2. Patient Sample Correlation to Reference Method

Concentrations measured by the illustrative assay system on the TDx®were compared with those measured by LC/MS on whole blood samples frompatients receiving everolimus therapy. Results of testing from tworeference laboratories are shown in FIG. 10 for kidney transplantpatients (data analyzed using Passing Bablock Linear RegressionAnalysis), comparing the illustrative assay system vs. LC/MS. Samplesranged on the TDx from 2.40 ng/mL to 33.17 ng/mL and are from 110individual patients. Similarly, results of testing from two referencelaboratories are shown in FIG. 11 for heart transplant patients (dataanalyzed using Passing Bablock Linear Regression Analysis), comparingthe illustrative assay system vs. LC/MS. Samples ranged on the TDx from2.06 ng/mL to 20.60 ng/mL and are from 62 different individual patients.The illustrative assay system shows a linear relationship betweenpercent dilution and recovery across the assay range.

Precision of the illustrative assay system was evaluated per NCCLSguidelines. Studies were performed by assaying each sample in duplicate,two runs per day for 20 non-consecutive days on a single analyzer, andcalibrating as needed. The results are shown below in Table 2. TABLE 2Precision study Level 1 Level 2 Level 3 Mean (ng/mL) 3.28 12.38 36.55Within Run SD (ng/mL) 0.35 0.73 3.13 Within Run % CV 11% 6% 9% Day toDay SD (ng/mL) 0.39 0.63 1.85 Day to Day % CV 12% 5% 5% Run to Run SD(ng/mL) 0.35 0.66 1.74 Run to Run % CV 11% 5% 5% Total SD (ng/mL) 0.631.17 4.03 Total % CV 19% 9% 11% 

3. Specificity—Cross-Reactivity

Studies were conducted to examine the cross-reactivity of the AntibodyReagent “A” to major everolimus metabolites. The compounds (in Table 3below) were added at 5 ng/mL to normal pooled human whole blood(containing everolimus spiked at the low end of the therapeutic range,approximately 4 ng/mL) and tested in the illustrative assay system. Thenormal human whole blood spiked with everolimus was tested as thecontrol.

Although tested at 5 ng/mL, lower metabolite concentrations would beexpected in actual patients on everolimus therapy. RAD SA, RAD and PSAhave been found in quantities <20% of parent drug in drug metabolismstudies of human subjects. The Hydroxyl-(24/25 OH RAD, 460H RAD)metabolites cross reactivity were not tested by spiked addition. TABLE 3Cross-reactivity: % cross-reactivity = ([measured everolimus with spikedmetabolite] − [measured control] ÷ [metabolite added]) × 100 CompoundConcentration Apparent % Cross Tested Tested (ng/mL) Conc. (ng/mL)reactivity RAD SA 5 0.26 5 RAD PSA 5 0.62 12

4. Specificity—Drug Interference

The illustrative assay system was tested against potentially interferingcompounds. The substances shown in Table 4, when added to human wholeblood (containing 12 ng/mL everolimus), had cross reactivity less than5% when tested at concentrations exceeding clinically relevant levels.TABLE 4 Drug Interference Drug Test Level (μg/mL) Acetaminophen 200N-Acetylprocainamide 120 Acyclovir 1000 Albuterol 0.18 Allopurinol 60Amikacin 150 Amphotericin B 100 Ascorbic Acid 30 Atenolol 40Azathioprine 10 Caffeine 100 Captopril 50 Carbamazepine 120 Cefaclor 230Chloramphenicol 250 Cimetidine 100 Ciprofloxacin 2500 Cyclosporin A 1Digoxin 10 Disopyramide 30 Erythromycin 200 Ethanol 3500 Folic Acid 0.01Furosemide 100 Ganciclovir 1000 Gentamicin 20 Glipzide 60 Glyburide 40Heparin 8000 U/L Hydralazine 32 Hydrochlorothiazide 40 Ibuprofen 400Insulin 400 μU/mL Intralipid 15000 Isoniazid 70 IsoproternolHydrochloride 0.06 Kanamycin 100 Ketoconazole 10 Labetalol 200 Lidocaine100 Lovastatin 4 Metformin HCl 5100 Metoclopramide 4 Misoprostol 0.015Morphine Sulfate 6 Mycophenolic Acid 250 Nadolol 333 Naproxen 1000Niacin 800 Nifedipine 120 Omeprazole 14 Penicillin G 100 Phenobarbital150 Phenytoin 100 Piperacillin 8 Prazosin 25 Prednisone 12 Prednisolone12 Primidone 100 Procainamide 25 Propanolol 0.5 Quinidine 100 Ranitidine200 Rifampin 50 Salicylic Acid 500 Spectinomycin 100 Sulfamethoxazole400 Tacrolimus 0.5 Theophyline 250 Tobramycin 20 Triamterene 600Trimethoprim 20 Valproic Acid 1000 Vancomycin 630 Verapamil 10Specificity—Interfering Substances

The following compounds, as shown in Table 5, when added to normal humanwhole blood containing everolimus at or below the low end of thetherapeutic range resulted in <10% error in quantitating everolimus bythe illustrative assay system. TABLE 5 Interfering Substances CompoundTested Concentration Tested Albumin 12 g/dL Bilirubin 20 mg/dLCholesterol 500 mg/dL Human Gamma Globulin 12 g/dL Rheumatoid Factor 500IU/mL Triglycerides 1,500 mg/dL

Hematocrits at 20% and 60%, resulted in <10% error in quantitatingeverolimus by the illustrative assay system.

5. Sensitivity

The Limit of Quantification (LOQ) of the illustrative assay system,defined as the lowest concentration in human whole blood at whichinter-assay CV between multiple replicates is ≦20%, is 2.00 ng/mL.

The lower limit of detection (LDD) for the illustrative assay system,defined as the lowest concentration that can be distinguished from zero,is 0.80 ng/mL.

It is understood that the above results are illustrative of oneembodiment of FPIA using polyclonal Antibody Reagent (RAD 822:BSA) andTracer Reagent (RAD 822:FAMCO-E). Other competitive assays within thescope of this invention may provide different performancecharacteristics.

All references cited herein are incorporated by reference as if fullyset forth.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

1. A competitive immunoassay to determine the presence of everolimus ina sample comprising an antibody capable of specifically bindingeverolimus, and an everolimus compound conjugated to a detectable label,wherein the conjugated everolimus compound is configured to compete withthe everolimus in the sample to bind with the antibody, and wherein thelabel provides a signal indicative of a concentration of everolimus inthe sample when the everolimus in the sample is present in therapeuticdrug monitoring concentrations.
 2. The competitive immunoassay of claim1 wherein the range of therapeutic drug concentrations includeseverolimus from about 3 to about 15 ng/ml.
 3. The competitiveimmunoassay of claim 1 wherein the signal is indicative of theconcentration of everolimus when everolimus is present from about 0 toabout 40 ng/ml.
 4. The competitive immunoassay of claim 1 wherein theantibody was produced using an antigen having the formula:

wherein n is 0 or 1; X is a linker chain comprising 3-10 carbon orhetero atoms, wherein the linker chain may be substituted orunsubstituted and may be straight or branched; Y is selected from thegroup consisting of —C(O)—, —NH—, —S—, —CH₂— and —O—; and Z is anantigenic carrier.
 5. The competitive immunoassay of claim 4 wherein theantibody was produced using an antigen having the formula:


6. The competitive immunoassay of claim 1 wherein the assay is selectedfrom the group consisting of an FPIA, an homogeneous microparticle(immunoturbidimetric) immunoassay, a cloned enzyme donor immunoassay(CEDIA), and a chemiluminescent heterogeneous immunoassay, and lateralflow immunoassay.
 7. The competitive immunoassay of claim 1 wherein theconjugated everolimus compound is a compound of the formula:

wherein X¹ is a linker chain comprising one or more atoms, each of whichmay be substituted or unsubstituted and may be branched or unbranched; Yis selected from the group consisting of —C(O)—, —NH—, —S—, —CH₂— and—O—; and Z is the label.
 8. The competitive immunoassay of claim 7wherein X¹ is —O—CH₂—, and Y is —C(O)—.
 9. The competitive immunoassayof claim 1 wherein the antibody exhibits strong binding and stronginhibition of greater than 50% displacement over an assay range witheverolimus.
 10. The competitive immunoassay of claim 1 provided as akit.
 11. The competitive immunoassay of claim 10, further comprising acalibrator.
 12. A method for determining the amount of everolimus in asample comprising mixing the sample with an antibody capable ofspecifically binding everolimus, and an everolimus compound conjugatedto a detectable label, wherein the conjugated everolimus compound isconfigured to compete with the everolimus in the sample to bind with theantibody, measuring a signal from the detectable label indicative of aconcentration of everolimus in the sample, and determining the amount ofeverolimus in the sample.
 13. The method of claim 14 wherein theeverolimus in the sample is present in therapeutic drug monitoringconcentrations.
 14. The method of claim 15 wherein the range oftherapeutic drug concentrations includes everolimus from about 3 toabout 15 ng/ml.
 15. The method of claim 13 wherein the antibody wasproduced using an antigen having the formula:

wherein n is 0 or 1; X is a linker chain comprising 3-10 carbon orhetero atoms, wherein the linker chain may be substituted orunsubstituted and may be straight or branched; Y is selected from thegroup consisting of —C(O)—, —NH—, —S—, —CH₂— and —O—; and Z is anantigenic carrier.
 16. The method of claim 13 wherein the sample is abody fluid.
 17. The method of claim 13 wherein the mixing step comprisesfirst mixing the antibody and the everolimus compound conjugated to thedetectable label and then adding the sample.
 18. The method of claim 13wherein the mixing step comprises first mixing the antibody and thesample and then adding the everolimus compound conjugated to thedetectable label.
 19. A compound having the following structure:

wherein n is 0 or 1; X is a linker chain comprising 3-10 carbon orhetero atoms, wherein the linker chain may be substituted orunsubstituted and may be straight or branched; Y is selected from thegroup consisting of —C(O)—, —NH—, —S—, —CH₂— and —O—; and Z is anantigenic carrier or a label.
 20. The compound of claim 19 wherein n=1and X is a linker group comprising 4-6 substituted or unsubstitutedstraight or branched chain carbon or hetero atoms.
 21. The compound ofclaim 20 wherein X is —CH₂—CH₂-CH₂—CH₂-CH₂— and Y is —C(O)—.
 22. Thecompound of claim 21 wherein Z is the antigenic carrier.
 23. An antibodyproduced using the compound of 22 that is capable of specific binding toeverolimus.
 24. The antibody of claim 23 wherein the antibody is amonoclonal antibody.
 25. The antibody of claim 23 wherein the antibodyis a polyclonal antibody:
 26. An immunoassay kit for detectingeverolimus in a sample comprising the antibody of claim 23, and adetectable label, wherein the label is configured to produce adetectable signal when the sample, the antibody, and the label are mixedtogether and everolimus is present in the sample.
 27. The immunoassay ofclaim 26 wherein the label is linked to the antibody or to a secondaryantibody having specificity for the antibody.
 28. The immunoassay ofclaim 26 wherein the label is linked to an everolimus molecule that isconfigured to compete with everolimus in the sample for binding with theantibody.